Progesterone (P4) is an essential hormone that elicits its actions at each level of the reproductive axis. In the uterus, P4 attenuates estradiol-induced epithelial cell proliferation and facilitates cellular differentiation in preparation for the establishment and maintenance of pregnancy. In the ovary, P4 acts directly on granulosa cells to inhibit mitosis and apoptosis despite these cells lacking expression of the classical progesterone receptor (PGR; i.e., PRA and PRB). P4 also promotes the viability and steroidogenic potential of luteal cells and stimulates both its own secretion and cholesterol synthesis. Many of the actions of P4 within the ovary and uterus are mediated by the PGR. However, not all of the actions of P4 can be explained by activation of PGR, since a number of cell lines that do not express this receptors, as well as Pgr null mice, are able to respond to P4. Our recent in vitro studies of ovarian and uterine cells have revealed that some actions of P4 such as granulosa/luteal cell viability, P4 synthesis and uterine stromal cell differentiation are mediated in part through the P4 binding protein, Progesterone Receptor Membrane Component-1 (PGRMC1). Through the use of conditional mutagenesis, we have established that Pgrmc1 is essential for normal fertility in that female mice lacking PGRMC1 display a subfertility phenotype and undergo premature ovarian failure (POF). This phenotype is also observed in some women where lower Pgrmc1 expression and haploinsufficiency associate with POF and polycystic ovarian syndrome. Pgrmc1 conditional KO (cKO) females also have uterine hyperplasia and develop both uterine and ovarian cysts. There is mounting evidence that PGRMC2 also plays an important role in female reproduction in that decreased PGRMC2 associates with advanced endometriosis in a primate model and may serve as a marker for the onset of parturition. These cumulative studies along with numerous in vitro studies clearly demonstrate that PGRMC1 and PGRMC2 play important and clinically relevant roles in regulating uterine and ovarian functions and that alteration in their expression results in the manifestation of disease states in the female reproductive system. In this grant we will now test our hypothesis that Pgrmc2 plays a fundamental role in uterine and ovarian physiology through the use of Pgrmc2 cKO mice. We also propose to assess fertility in female mice that are deficient in both Pgrmc1 and Pgrmc2 (double cKO mice). Breeding trials will initially be completed to determine fertility status of cKO mice. Other parameters to be evaluated in Aim 1 include uterine decidualization, ovulation, ovarian steroidogenesis, oocyte quality, and embryo implantation. In Aim 2, we will evaluate the importance of Pgrmc2 in mediating the anti-proliferative and anti-apoptotic actions of P4 in the ovary, as well as in the estrogen stimulated uterus. Finally, it is clear from studies of PGR mutant mice that PGR is not the sole mediator of P4-induced gene transcription. In Aim 2, we plan to also evaluate P4-induced transcriptional responses in the uterus and ovary of Pgrmc1/2 double cKO mice using RNA-seq. The successful completion of the proposed studies will provide compelling evident to support a role for PGRMC2 in female reproductive physiology. Establishing PGRMC2 as a mediator of specific uterine and ovarian functions will allow for the development of a new and selective class of P4 antagonists/agonists that target the PGRMC family.